TY - JOUR
T1 - Integrin αIIbβ3
T2 - ligand interactions are linked to binding-site remodeling
AU - Hantgan, Roy R.
AU - Stahle, Mary C.
AU - Connor, John H.
AU - Horita, David A.
AU - Rocco, Mattia
AU - Mclane, Marya
AU - Yakovlev, Sergiy
AU - Medved, Leonid
PY - 2006
Y1 - 2006
N2 - This study tested the hypothesis that high-affinity binding of macromolecular ligands to the αIIbβ3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions be tween αIIbβ3 and two model ligands - echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's γ-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site - were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the αIIb/β3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming - i.e., ectodomain conformational changes and oligomerization induced by incubation at 35°C with the ligand-mimetic peptide cHarGD - promotes complex formation with fibrinogen's γ-module. We also observed that the γ-module's flexible carboxy terminus was not required for αIIbβ3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers. Published by Cold Spring Harbor Laboratory Press.
AB - This study tested the hypothesis that high-affinity binding of macromolecular ligands to the αIIbβ3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions be tween αIIbβ3 and two model ligands - echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's γ-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site - were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the αIIb/β3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming - i.e., ectodomain conformational changes and oligomerization induced by incubation at 35°C with the ligand-mimetic peptide cHarGD - promotes complex formation with fibrinogen's γ-module. We also observed that the γ-module's flexible carboxy terminus was not required for αIIbβ3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers. Published by Cold Spring Harbor Laboratory Press.
KW - Analytical ultracentrifugation
KW - Conformation
KW - Fluorescence anisotropy
KW - Integrins
KW - Ligands
KW - Molecular modeling
KW - Solid phase binding
UR - http://www.scopus.com/inward/record.url?scp=33746571991&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33746571991&partnerID=8YFLogxK
U2 - 10.1110/ps.052049506
DO - 10.1110/ps.052049506
M3 - Article
C2 - 16877710
AN - SCOPUS:33746571991
VL - 15
SP - 1893
EP - 1906
JO - Protein Science
JF - Protein Science
SN - 0961-8368
IS - 8
ER -